Human arsenite-resistance protein

ABSTRACT

The invention provides a human arsenite-resistance protein (NITE) and polynucleotides which identify and encode NITE. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of NITE.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman arsenite-resistance protein and to the use of these sequences inthe diagnosis, treatment, and prevention of cancer, developmentaldisorders, reproductive disorders, and autoimmune/inflammatorydisorders.

BACKGROUND OF THE INVENTION

Arsenic is a toxin and carcinogen found in water, soil, food, and air.The natural source of arsenic is from weathering of arsenic-richgeologic formations. Arsenic also derives from human agricultural andindustrial sources. Arsenic is used in pesticides, wood preservatives,and manufacturing of glass, paper, and semiconductors. This toxin isreleased into the environment during metal smelting, mining, fossil fuelburning, and incineration. Arsine gas has been used as a chemicalwarfare agent. Inorganic forms of arsenic, including arsenite(trivalent) and arsenate (pentavalent), are more toxic than organicforms, including methylarsonate and dimethylarsinate. Methyltransferasesin humans and animals methylate inorganic arsenic, facilitating itsexcretion. (Shalat, S. L. et al. (1996) J. Toxicol. Env. Health48:253-272; Jager, J. W. and Ostrosky-Wegman, P. (1997) Mutat. Res.386:181-184; and Graeme, K. A. and Pollack, Jr., C. V. (1998) J. Emerg.Med. 16:45-56.)

Acute arsenic poisoning can be fatal, killing by impairment of cellularrespiration in the case of arsenite and arsenate, or by hemolysis in thecase of arsine gas. The effects of chronic low arsenic exposure are ofinterest due to increasing pollution of water, air, and food witharsenic from industrial and agricultural sources. Chronic arsenicexposure is associated with skin and lung cancers in humans, but not inanimals. Arsenite causes chromosomal aberrations, sister chromatidexchanges, and cell transformation, and acts as a comutagen withultraviolet light and the alkylating agent N-methyl-N-nitrosourea.(Wang, Z. and Rossman, T. G. (1993) Toxicol. Appl. Pharmacol.118:80-86.) Arsenic can cross the placenta and selectively accumulate inthe fetal neuroepithelium during early embryogenesis. Maternal orpaternal exposure to arsenic is associated with spontaneous abortion,stillbirth, and congenital malformations and developmental impairment inthe infant. Neural tube defects such as dysraphic spina bifida andanencephaly are predominant. (Shalat, supra.) Other effects in humansinclude peripheral neuropathy, microangiopathy, bone marrow and immunesuppression, and cardiovascular disease. (Jager, supra; and Shalat,supra.)

Arsenic compounds exert their effects by a variety of means. Arsenitereacts with thiol groups in macromolecules such as proteins and nucleicacids. Glycolytic and β-oxidation enzymes are inhibited by thisreaction, which may cause increased and unregulated cell death. Thiscell death may cause developmental defects because reduced cellproliferation is associated with neural tube defects in mice. (Shalat,supra.) Inorganic arsenic impairs the assembly and disassembly ofmicrotubules and may interfere with mitotic spindle formation, embryoniccell division, and neural tube formation. Arsenite reaction with thethiols present in histones and nucleic acids may cause the chromosomaldamage associated with arsenic exposure. Arsenate can substitute forphosphate in enzymatic reactions, forming acyl arsenates. However, acylarsenates are less stable than the corresponding phosphates anddecompose rapidly, inhibiting glycolytic and oxidative phosphorylationenzymes. (Wang, Z. et al. (1996) Toxicol. Appl. Pharmacol. 137:112-119.)

To understand the mechanism of arsenic toxicity and carcinogenicity,studies involving arsenite resistance in cultured Chinese hamster andhuman cells have been undertaken. Hamster cells become tolerant to toxiclevels of arsenite after pretreatment with low levels. Stablearsenite-resistant hamster cells have also been selected. Expressioncloning of arsenite-resistance genes from Chinese hamster identified twogenes, ars1 and ars2. Ars1 is similar to ubiquitin, while ars2 is novel.(Rossman, T. G. and Wang, Z., unpublished; Wang (1993) supra; andRossman, T. G. et al. (1997) Mutat. Res. 386:307-314.) Human cells couldnot be induced to become arsenite-tolerant under the conditions used forhamster cells. This result may explain why humans develop tumors afterarsenite exposure, while rodents do not. The researchers concluded thathumans may be unable to induce one or more protective proteins inresponse to arsenite. Possible mechanisms for arsenite resistanceinclude upregulation of an arsenic efflux pump, increased rates oftransformation of arsenite to arsenate and organic forms, increasedbinding to proteins, and elevation of glutathione levels and glutathioneS-transferase activity. (Wang, (1993) supra; and Rossman, (1997) supra.)

The discovery of a new human arsenite-resistance protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are usefull in the diagnosis, treatment, andprevention of cancer, developmental disorders, reproductive disorders,and autoimmune/inflammatory disorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery of a new humanarsenite-resistance protein (NITE), the polynucleotides encoding NITE,and the use of these compositions for the diagnosis, treatment, orprevention of cancer, developmental disorders, reproductive disorders,and autoimmune/inflammatory disorders.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant havingat least 90% amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 90% polynucleotide sequence identity to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

The invention further provides an isolated and purified polynucleotidewhich hybridizes under stringent conditions to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO: 1 or a fragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2, and an isolated and purified polynucleotide variant havingat least 90% polynucleotide sequence identity to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides an isolated and purifiedpolynucleotide having a sequence complementary to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2 .

The invention further provides an expression vector comprising at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcomprising an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the sequence of SEQ ID NO:1 ora fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of a pharmaceutical composition comprisingsubstantially purified polypeptide having the amino acid sequence of SEQID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing adevelopmental disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing anautoimmune/inflammatory disorder, the method comprising administering toa subject in need of such treatment an effective amount of apharmaceutical composition comprising substantially purified polypeptidehaving the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ IDNO:1.

The invention also provides a method for detecting a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1or a fragment of SEQ ID NO:1 in a biological sample containing nucleicacids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to atleast one of the nucleic acids of the biological sample, thereby forminga hybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in thebiological sample. In one aspect, the nucleic acids of the biologicalsample are amplified by the polymerase chain reaction prior to thehybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of NITE. The alignment was producedusing MacDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd.,San Bruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence alignment between NITE(Incyte Clone number 2257452; SEQ ID NO:1), and Chinese hamsterarsenite-resistance protein (ars2) (GI 1127863; SEQ ID NO:3), producedusing the multisequence alignment program of LASERGENE™ software(DNASTAR Inc, Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

DEFINITIONS

"NITE," as used herein, refers to the amino acid sequences ofsubstantially purified NITE obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to NITE, increases or prolongs the duration of the effect of NITE.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of NITE.

An "allelic variant," as this term is used herein, is an alternativeform of the gene encoding NITE. Allelic variants may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

"Altered" nucleic acid sequences encoding NITE, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as NITE ora polypeptide with at least one functional characteristic of NITE.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding NITE, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding NITE. The encoded proteinmay also be "altered," and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent NITE. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of NITE is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments," "immunogenic fragments," or"antigenic fragments" refer to fragments of NITE which are preferablyabout 5 to about 15 amino acids in length, most preferably 14 aminoacids, and which retain some biological activity or immunologicalactivity of NITE. Where "amino acid sequence" is recited herein to referto an amino acid sequence of a naturally occurring protein molecule,"amino acid sequence" and like terms are not meant to limit the aminoacid sequence to the complete native amino acid sequence associated withthe recited protein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer. a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp.1-5.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to NITE, decreases the amount or the duration of the effectof the biological or immunological activity of NITE. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of NITE.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindNITE polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the "sense"strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation "negative" canrefer to the antisense strand, and the designation "positive" can referto the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic NITE, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides by base pairing. For example, thesequence "A-G-T" binds to the complementary sequence "T-C-A."Complementarity between two single-stranded molecules may be "partial,"such that only some of the nucleic acids bind, or it may be "complete,"such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding NITE orfragments of NITE may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts, e.g., NaCl,detergents, e.g.,sodium dodecyl sulfate (SDS), and other components,e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.

"Consensus sequence," as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW™ Fragment Assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding NITE, byNorthern analysis is indicative of the presence of nucleic acidsencoding NITE in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding NITE.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term "similarity," as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word "identity" may substitute for the word "similarity." Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as "substantially similar." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign™ program (DNASTAR, Inc.,Madison Wis.). The MegAlign™ program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀ t or R₀ t analysis) or formed betweenone nucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

"Immune response" can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term "microarray," as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms "element" or "array element" as used herein in a microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term "modulate," as it appears herein, refers to a change in theactivity of NITE. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of NITE.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, "fragments" refersto those nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms "operably associated" or "operably linked," as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

The term "oligonucleotide," as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms "amplimer,""primer," "oligomer," and "probe," as these terms are commonly definedin the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anti cancer Drug Des. 8:53-63.)

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding NITE,or fragments thereof, or NITE itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotides and the claimedpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent (e.g., formamide),temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and most preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and most preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/mldenatured salmon sperm DNA (ssDNA). In a most preferred embodiment,hybridization will occur at 42° C. in 250 mM NaCl, 25 mM trisodiumcitrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variationson these conditions will be readily apparent to those skilled in theart.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A "variant" of NITE, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, for27 example, LASERGENE™ software.

THE INVENTION

The invention is based on the discovery of a new humanarsenite-resistance protein (NITE), the polynucleotides encoding NITE,and the use of these compositions for the diagnosis, treatment, orprevention of cancer, developmental disorders, reproductive disorders,and autoimmune/inflammatory disorders.

Nucleic acids encoding the NITE of the present invention were firstidentified in Incyte Clone 2257452 from the ovarian tumor cDNA library(OVARTUT01) using a computer search, e.g., BLAST, for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 640360 (BRSTNOT03), 841402 (PROSTUT05), 1595967 (BRAINOT14),2017563 (THP1NOT01), and 2257452 (OVARTUT01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,and 1D. NITE is 429 amino acids in length and has two potentialamidation sites at residues M241 and S271; one potential N-glycosylationsite at N113; seven potential casein kinase II phosphorylation sites atresidues T83, S123, S208, T218, S228, S232, and S234; three potentialprotein kinase C phosphorylation sites at T54, S228, and S271; and onepotential cell attachment sequence at R408GD. As shown in FIGS. 2A and2B, NITE has chemical and structural similarity with Chinese hamsterarsenite-resistance protein (ars2) (GI1127863; SEQ ID NO:3). Inparticular, NITE and ars2 protein share 70% identity. A fragment of SEQID NO:2 from about nucleotide 57 to about nucleotide 80 is useful, forexample, as a hybridization probe. Northern analysis shows theexpression of this sequence in various libraries, at least 44% of whichare immortalized or cancerous, at least 40% of which involve immuneresponse, and at least 17% involve fetal or proliferating cells. Ofparticular note is the expression of NITE in male reproductive, femalereproductive, hematopoietic/immune, gastrointestinal, cardiovascular,nervous, and developmental tissues.

The invention also encompasses NITE variants. A preferred NITE variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe NITE amino acid sequence, and which contains at least one functionalor structural characteristic of NITE.

The invention also encompasses polynucleotides which encode NITE. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an NITE.

The invention also encompasses a variant of a polynucleotide sequenceencoding NITE. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding NITE. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of NITE.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding NITE, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring NITE, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode NITE and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring NITE under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding NITE or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding NITE and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeNITE and NITE derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents well known in the art. Moreover, synthetic chemistry may beused to introduce mutations into a sequence encoding NITE or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, Sequenase® (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), therrnostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE™ Amplification System(GIBco BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.)and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding NITE may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder® libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO™ 4.06 Primer Analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5'regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5' non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., Genotyper™ and Sequence Navigator™, PerkinElmer), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode NITE may be cloned in recombinant DNAmolecules that direct expression of NITE, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express NITE.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter NITE-encodingsequences for a variety of purposes including, but not limited to,modification of the cloning, processing, and/or expression of the geneproduct. DNA shuffling by random fragmentation and PCR reassembly ofgene fragments and synthetic oligonucleotides may be used to engineerthe nucleotide sequences. For example, oligonucleotide-mediatedsite-directed mutagenesis may be used to introduce mutations that createnew restriction sites, alter glycosylation patterns, change codonpreference, produce splice variants, and so forth.

In another embodiment, sequences encoding NITE may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, NITE itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A Peptide Synthesizer (Perkin Elmer).Additionally, the amino acid sequence of NITE, or any part thereof, maybe altered during direct synthesis and/or combined with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins. Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active NITE, the nucleotide sequencesencoding NITE or derivatives thereof may be inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcriptional and translational control of the inserted codingsequence in a suitable host. These elements include regulatorysequences, such as enhancers, constitutive and inducible promoters, and5' and 3' untranslated regions in the vector and in polynucleotidesequences encoding NITE. Such elements may vary in their strength andspecificity. Specific initiation signals may also be used to achievemore efficient translation of sequences encoding NITE. Such signalsinclude the ATG initiation codon and adjacent sequences, e.g. the Kozaksequence. In cases where sequences encoding NITE and its initiationcodon and upstream regulatory sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding NITE andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding NITE. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding NITE. For example, routine cloning, subcloning, and propagationof polynucleotide sequences encoding NITE can be achieved using amultifunctional E. coli vector such as Bluescript® (Stratagene) orpSport1™ plasmid (GImco BRL). Ligation of sequences encoding NITE intothe vector's multiple cloning site disrupts the lacZ gene, allowing acalorimetric screening procedure for identification of transformedbacteria containing recombinant molecules. In addition, these vectorsmay be useful for in vitro transcription, dideoxy sequencing, singlestrand rescue with helper phage, and creation of nested deletions in thecloned sequence. (See, e.g., Van Heeke, G. and S. M. Schuster (1989) J.Biol. Chem. 264:5503-5509.) When large quantities of NITE are needed,e.g. for the production of antibodies, vectors which direct high levelexpression of NITE may be used. For example, vectors containing thestrong, inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of NITE. A number ofvectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) MethodsEnzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology12:181-184.)

Plant systems may also be used for expression of NITE. Transcription ofsequences encoding NITE may be driven viral promoters, e.g., the 35S and19S promoters of CaMV used alone or in combination with the omega leadersequence from TMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.)Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used. (See, e.g., Coruzzi, G. et al. (1984)EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; andWinter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. (See, e.g., Hobbs, S.or Murry, L. E. in McGraw Hill Yearbook of Science and Technology (1992)McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding NITE may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses NITE in host cells. (See, e.g., S Logan, J. and T. Shenk(1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems,expression of NITE in cell lines is preferred. For example, sequencesencoding NITE can be transformed into cell lines using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for about 1 to 2 days in enriched media before beingswitched to selective media. The purpose of the selectable marker is toconfer resistance to a selective agent, and its presence allows growthand recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB and hisD, which altercellular requirements for metabolites. (See, e.g., Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech,Palo Alto, Calif.), βglucuronidase and its substrate β-D-glucuronoside,or luciferase and its substrate luciferin may be used. These markers canbe used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingNITE is inserted within a marker gene sequence, transformed cellscontaining sequences encoding NITE can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding NITE under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingNITE and that express NITE may be identified by a variety of proceduresknown to those of skill in the art. These procedures include, but arenot limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification,and protein bioassay or immunoassay techniques which include membrane,solution, or chip based technologies for the detection and/orquantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression of NITEusing either specific polyclonal or monoclonal antibodies are known inthe art. Examples of such techniques include enzyme-linked immunosorbentassays (ELISAs), radioimmunoassays (RIAs), and fluorescence activatedcell sorting (FACS). A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering epitopes on NITEis preferred, but a competitive binding assay may be employed. These andother assays are well known in the art. (See, e.g., Hampton, R. et al.(1990) Serological Methods, a Laboratory Manual, APS Press, St Paul,Minn., Section IV; Coligan, J. E. et al. (1997 and periodic supplements)Current Protocols in Immunology, Greene Pub. Associates andWiley-Interscience, New York, N.Y.; and Maddox, D. E. et al. (1983) J.Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding NITE includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding NITE,or any fragments thereof, may be cloned into a vector for the productionof an MRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Minn.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding NITE may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeNITE may be designed to contain signal sequences which direct secretionof NITE through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC, Bethesda, Md.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding NITE may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric NITEprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of NITE activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these ealso be engineerusion protein may also be engineered tocontain a proteolytic cleavage site located between the NITE encodingsequence and the heterologous protein sequence, so that NITE may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeled NITEmay be achieved in vitro using the TNT™rabbit reticulocyte lysate orwheat germ extract systems (Promega, Madison, Wis.). These systemscouple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵ S-methionine.

Fragments of NITE may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the Applied Biosystems 431A Peptide Synthesizer(Perkin Elmer). Various fragments of NITE may be synthesized separatelyand then combined to produce the full length molecule.

THERAPEUTICS

Chemical and structural similarity exists between NITE andarsenite-resistance protein (ars2) from Chinese hamster (GI 1127863). Inaddition, NITE is expressed in cancerous, inflamed, fetal orproliferating, reproductive, hematopoietic/immune, and developmentaltissues. Therefore, NITE appears to play a role in cancer, developmentaldisorders, reproductive disorders, and autoimmune/inflammatorydisorders.

Therefore, in one embodiment, NITE or a fragment or derivative thereofmay be administered to a subject to treat or prevent a cancer. Suchcancers can include, but are not limited to, adenocarcinoma, leukemia,lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, inparticular, cancers of the adrenal gland, bladder, bone, bone marrow,brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract,heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis,prostate, salivary glands, skin, spleen, testis, thymus, thyroid, anduterus.

In another embodiment, a vector capable of expressing NITE or a fragmentor derivative thereof may be administered to a subject to treat orprevent a cancer including, but not limited to, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified NITE in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a cancer including, but not limited to, those provided above.

In still another embodiment, an agonist which modulates the activity ofNITE may be administered to a subject to treat or prevent a cancerincluding, but not limited to, those listed above.

In another embodiment, NITE or a fragment or derivative thereof may beadministered to a subject to treat or prevent a developmental disorder.Such developmental disorders can include, but are not limited to, renaltubular acidosis, anemia, Cushing's syndrome, achondroplastic dwarfism,Duchenne and Becker muscular dystrophy, epilepsy, gonadal dysgenesis,WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, andmental retardation), Smith-Magenis syndrome, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spina bifida, anencephaly,craniorachischisis, congenital glaucoma, cataract, and sensorineuralhearing loss.

In another embodiment, a vector capable of expressing NITE or a fragmentor derivative thereof may be administered to a subject to treat orprevent a developmental disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified NITE in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a developmental disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofNITE may be administered to a subject to treat or prevent adevelopmental disorder including, but not limited to, those listedabove.

In another embodiment, NITE or a fragment or derivative thereof may beadministered to a subject to treat or prevent a reproductive disorder.Such reproductive disorders can include, but are not limited to,disorders of prolactin production; infertility, including tubal disease,ovulatory defects, and endometriosis; disruptions of the estrous cycle,disruptions of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, uterinefibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea;disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, Peyronie's disease, carcinoma of the male breast, andgynecomastia.

In another embodiment, a vector capable of expressing NITE or a fragmentor derivative thereof may be administered to a subject to treat orprevent a reproductive disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified NITE in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a reproductive disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofNITE may be administered to a subject to treat or prevent a reproductivedisorder including, but not limited to, those listed above.

In another embodiment, NITE or a fragment or derivative thereof may beadministered to a subject to treat or prevent an autoimmune/inflammatorydisorder. Such autoimmune/inflammatory disorders can include, but arenot limited to, acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowelsyndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome,gren's syndrome, systemicanaphylaxis, systemic lupus erythematosus, systemic sclerosis,thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome,complications of cancer, hemodialysis, and extracorporeal circulation,viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections, and trauma.

In another embodiment, a vector capable of expressing NITE or a fragmentor derivative thereof may be administered to a subject to treat orprevent an autoimmune/inflammatory disorder including, but not limitedto, those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified NITE in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent an autoimmune/inflanunatory disorder including, but not limitedto, those provided above.

In still another embodiment, an agonist which modulates the activity ofNITE may be administered to a subject to treat or prevent anautoimmune/inflammatory disorder including, but not limited to, thoselisted above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of NITE may be produced using methods which are generallyknown in the art. In particular, purified NITE may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind NITE. Antibodies to NITE may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith NITE or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to NITE have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of NITE amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to NITE may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce NITE-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci.86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for NITE mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between NITE and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering NITE epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingNITE, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingNITE may be used in situations in which it would be desirable to blockthe transcription of the MRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding NITE. Thus,complementary molecules or fragments may be used to modulate NITEactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding NITE.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding NITE. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

Genes encoding NITE can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding NITE. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingNITE. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof MRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingNITE.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include, techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding NITE. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the molecule,or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of NITE,antibodies to NITE, and mimetics, agonists, antagonists, or inhibitorsof NITE. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances whcarboxymethyl cellulossity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of NITE, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example NITE or fragments thereof, antibodies of NITE,and agonists, antagonists or inhibitors of NITE, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED₅₀ LD₅₀ ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind NITE may beused for the diagnosis of disorders characterized by expression of NITE,or in assays to monitor patients being treated with NITE or agonists,antagonists, or inhibitors of NITE. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for NITE include methods which utilizethe antibody and a label to detect NITE in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring NITE, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of NITE expression. Normal or standard values for NITEexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toNITE under conditions suitable for complex formation The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of NITE expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingNITE may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofNITE may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of NITE, and tomonitor regulation of NITE levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding NITE or closely related molecules may be used to identifynucleic acid sequences which encode NITE. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding NITE, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the NITEencoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ ID NO:2 orfrom genomic sequences including promoters, enhancers, and introns ofthe NITE gene.

Means for producing specific hybridization probes for DNAs encoding NITEinclude the cloning of polynucleotide sequences encoding NITE or NITEderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² P or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding NITE may be used for the diagnosis ofa disorder associated with expression of NITE. Examples of such adisorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; developmental disorderssuch as renal tubular acidosis, anemia, Cushing's syndrome,achondroplastic dwarfism, Duchenne and Becker muscular dystrophy,epilepsy, gonadal dysgenesis, WAGR syndrome (Wilms' tumor, aniridia,genitourinary abnormalities, and mental retardation), Smith-Magenissyndrome, myelodysplastic syndrome, hereditary mucoepithelial dysplasia,hereditary keratodermas, hereditary neuropathies such asCharcot-Marie-Tooth disease and neurofibromatosis, hypothyroidism,hydrocephalus, seizure disorders such as Syndenham's chorea and cerebralpalsy, spina bifida, anencephaly, craniorachischisis, congenitalglaucoma, cataract, and sensorineural hearing loss; reproductivedisorders such as disorders of prolactin production; infertility,including tubal disease, ovulatory defects, and endometriosis;disruptions of the estrous cycle, disruptions of the menstrual cycle,polycystic ovary syndrome, ovarian hyperstimulation syndrome,endometrial and ovarian tumors, uterine fibroids, autoimmune disorders,ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia; andautoimmune/inflammatory disorders such as acquired immunodeficiencysyndrome (AIDS), Addison's disease, adult respiratory distress syndrome,allergies, ankylosing spondylitis, amyloidosis, anemia, asthma,atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis,bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopicdermatitis, dermatomyositis, diabetes mellitus, emphysema, episodiclymphopenia with lymphocytotoxins, erythroblastosis fetalis, erythemanodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,irritable bowel syndrome, multiple sclerosis, myasthenia gravis,myocardial or pericardial inflammation, osteoarthritis, osteoporosis,pancreatitis, polymyositis, psoriasis, Reiter's syndrome,rheumatoidgren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. Thepolynucleotide sequences encoding NITE may be used in Southern orNorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered NITEexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding NITE may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingNITE may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding NITE in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of NITE , a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding NITE, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding NITE may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding NITE, or a fragment of a polynucleotide complementary to thepolynucleotide encoding NITE, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of NITEinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or calorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a nicroarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingNITE may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding NITE on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q 22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, NITE, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between NITEand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with NITE, orfragments thereof, and washed. Bound NITE is then detected by methodswell known in the art. Purified NITE can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding NITE specificallycompete with a test compound for binding NITE. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with NITE.

In additional embodiments, the nucleotide sequences which encode NITEmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. OVARTUTOI cDNA Library Construction

The OVARTUTOI cDNA library was constructed from tumorous ovarian tissueobtained from a 43-year-old Caucasian female during a bilateralsalpingo-oophorectomy. Pathology indicated a grade 2 cystadenocarcinomaof the left ovary forming a multilocular solid and cystic mass with asmooth external surface. The frozen tissue was homogenized and lysedusing a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments,Westbury, N.Y.) in guanidinium isothiocyanate solution. The lysate wascentrifuged over a 5.7M CsCl cushion using an Beckman SW28 rotor in aBeckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at25,000 rpm at ambient temperature. The RNA was extracted with acidphenol pH 4.7, precipitated using 0.3M sodium acetate and 2.5 volumes ofethanol, resuspended in RNAse-free water, and treated with DNase at 37°C. The RNA extraction and precipitation were repeated as before. ThemRNA was isolated with the Qiagen Oligotex kit (QIAGEN, Inc.,Chatsworth, Calif.) and used to construct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperScript™ Plasmid System for cDNA synthesis and plasmid cloning(Catalog #18248-013, GIBco BRL). The cDNAs were fractionated on aSepharose CL4B column (Catalog #275105-01, Pharmacia), and those cDNAsexceeding 400 bp were ligated into pSport I (Catalog #15382-013, GiBcoBRL). The plasmid pSport I was subsequently transformed into DH5α™competent cells (Catalog #18258-012, GImco BRL).

II. Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 plasmid kit (Catalog #26173, QIAGEN, Inc.). This kit enabled thesimultaneous purification of 96 samples in a 96-well block usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes: 1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, GImco BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) after inoculation, the cultures wereincubated for 19 hours and at the end of incubation, the cells werelysed with 0.3 ml of lysis buffer; and 3) following isopropanolprecipitation, the plasmid DNA pellet was resuspended in 0.1 ml ofdistilled water. After the last step in the protocol, samples weretransferred to a 96-well block for storage at 4° C.

The cDNAs were sequenced by the method of Sanger et al, (1975, J. Mol.Biol. 94:441f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems, andthe reading frame was determined.

III. Similarity Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofsimilarity using BLAST (Basic Local Alignment Search Tool). (See, e.g.,Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.(1990) J. Mol. Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms couldhave been used when dealing with primary sequence patterns and secondarystructure gap penalties. (See, e.g., Smith, T. et al. (1992) ProteinEngineering 5:35-51.) The sequences disclosed in this application havelengths of at least 49 nucleotides and have no more than 12% uncalledbases (where N is recorded rather than A, C, G, or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻²⁵ for nucleotides and 10⁻⁸ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for similarity.

Additionally, sequences identified from cDNA libraries may be analyzedto identify those gene sequences encoding conserved protein motifs usingan appropriate analysis program, e.g., BLOCKS. BLOCKS is a weightedmatrix analysis algorithm based on short amino acid segments, or blocks,compiled from the PROSITE database. (Bairoch, A. et al. (1997) NucleicAcids Res. 25:217-221.) The BLOCKS algorithm is useful for classifyinggenes with unknown functions. (Henikoff S. And Henikoff G. J., NucleicAcids Research (1991) 19:6565-6572.) Blocks, which are 3-60 amino acidsin length, correspond to the most highly conserved regions of proteins.The BLOCKS algorithm compares a query sequence with a weighted scoringmatrix of blocks in the BLOCKS database. Blocks in the BLOCKS databaseare calibrated against protein sequences with known functions from theSWISS-PROT database to determine the stochastic distribution of matches.Similar databases such as PRINTS, a protein fingerprint database, arealso searchable using the BLOCKS algorithm. (Attwood, T. K. et al.(1997) J. Chem. Inf. Comput. Sci. 37:417-424.) PRINTS is based onnon-redundant sequences obtained from sources such as SWISS-PROT,GenBank, PIR, and NRL-3D.

The BLOCKS algorithm searches for matches between a query sequence andthe BLOCKS or PRINTS database and evaluates the statistical significanceof any matches found. Matches from a BLOCKS or PRINTS search can beevaluated on two levels, local similarity and global similarity. Thedegree of local similarity is measured by scores, and the extent ofglobal similarity is measured by score ranking and probability values. Ascore of 1000 or greater for a BLOCKS match of highest ranking indicatesthat the match falls within the 0.5 percentile level of false positiveswhen the matched block is calibrated against SWISS-PROT. Likewise, aprobability value of less than 1.0×10⁻³ indicates that the match wouldoccur by chance no more than one time in every 1000 searches. Only thosematches with a cutoff score of 1000 or greater and a cutoff probabilityvalue of 1.0×1 or less are considered in the functional analyses of theprotein sequences in the Sequence Listing.

Nucleic and amino acid sequences of the Sequence Listing may also beanalyzed using PFAM. PFAM is a Hidden Markov Model (HMM) based protocoluseful in protein family searching. HMM is a probabilistic approachwhich analyzes consensus primary structures of gene families. (See,e.g., Eddy, S. R. (1996) Curr. Opin. Str. Biol. 6:361-365.)

The PFAM database contains protein sequences of 527 protein familiesgathered from publicly available sources, e.g., SWISS-PROT and PROSITE.PFAM searches for well characterized protein domain families using twohigh-quality alignment routines, seed alignment and full alignment.(See, e.g., Sonnhammer, E. L. L. et al. (1997) Proteins 28:405-420.) Theseed alignment utilizes the hmmls program, a program that searches forlocal matches, and a non-redundant set of the PFAM database. The fullalignment utilizes the hmmfs program, a program that searches formultiple fragments in long sequences, e.g., repeats and motifs, and allsequences in the PFAM database. A result or score of 100 "bits" cansignify that it is 2¹⁰⁰ -fold more likely that the sequence is a truematch to the model or comparison sequence. Cutoff scores which rangefrom 10 to 50 bits are generally used for individual protein familiesusing the SWISS-PROT sequences as model or comparison sequences.

Two other algorithms, SIGPEPT and TM, both based on the HMM algorithmdescribed above (see, e.g., Eddy, supra; and Sonnhammer, supra),identify potential signal sequences and transmembrane domains,respectively. SIGPEPT was created using protein sequences having signalsequence annotations derived from SWISS-PROT. It contains about 1413non-redundant signal sequences ranging in length from 14 to 36 aminoacid residues. TM was created similarly using transmembrane domainannotations. It contains about 453 non-redundant transmembrane sequencesencompassing 1579 transmembrane domain segments. Suitable HMM modelswere constructed using the above sequences and were refined with knownSWISS-PROT signal peptide sequences or transmembrane domain sequencesuntil a high correlation coefficient, a measurement of the correctnessof the analysis, was obtained. Using the protein sequences from theSWISS-PROT database as a test set, a cutoff score of 11 bits, asdetermined above, correlated with 91-94% true-positives and about 4.1%false-positives, yielding a correlation coefficient of about 0.87-0.90for SIGPEPT. A score of 11 bits for TM will typically give the followingresults: 75% true positives; 1.72% false positives; and a correlationcoefficient of 0.76. Each search evaluates the statistical significanceof any matches found and reports only those matches that score at least11 bits.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ™ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar.

The basis of the search is the product score, which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of Northern analysis are reported as a list of libraries inwhich the transcript encoding NITE occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of NITE Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 2257452 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence "outward" generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGOT 4.06(National Biosciences, Plymouth, Minn.), or another appropriate program,to be about 22 to 30 nucleotides in length, to have a GC content ofabout 50% or more, and to anneal to the target sequence at temperaturesof about 68° C. to about 72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations wasavoided.

Selected human cDNA libraries (GIBco BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR™ kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier Thermal Cycler(PTC200; M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation)

Step 2 65° C. for 1 min

Step 3 68° C. for 6 min

Step 4 94° C. for 15 sec

Step 5 65° C. for 1 min

Step 6 68° C. for 7 min

Step 7 Repeat steps 4 through 6 for an additional 15 cycles

Step 8 94° C. for 15 sec

Step 9 65° C. for 1 min

Step 10 68° C. for 7:15 min

Step 11 Repeat steps 8 through 10 for an additional 12 cycles

Step 12 72° C. for 8 min

Step 13 4° C. (and holding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK™ (QIAGEN Inc.), and trimmed ofoverhangs using Kienow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin (2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec

Step 2 94° C. for 20 sec

Step 3 55° C. for 30 sec

Step 4 72° C. for 90 sec

Step 5 Repeat steps 2 through 4 for an additional 29 cycles

Step 6 72° C. for 180 sec

Step 7 4° C. (and holding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genornic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO™ 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of γ-³² P! adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN®, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a Sephadex™ G-25 superfine size exclusiondextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots to film for severalhours, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE™. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the NITE-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring NITE. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of NITE. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the NITE-encoding transcript.

IX. Expression of NITE

Expression and purification of NITE is achieved using bacterial orvirus-based expression systems. For expression of NITE in bacteria, cDNAis subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21 (DE3). Antibiotic resistant bacteria express NITE uponinduction with isopropyl beta-D-thiogalactopyranoside (IPTG). Expressionof NITE in eukaryotic cells is achieved by infecting insect or mammaliancell lines with recombinant Autographica californica nuclearpolyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding NITE by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)Hum. Gene Ther. 7:1937-1945.)

In most expression systems, NITE is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (Pharmacia,Piscataway, N.J.). Following purification, the GST moiety can beproteolytically cleaved from NITE at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified NITE obtained by these methods canbe used directly in the following activity assay.

X. Demonstration of NITE Activity

NITE is labeled with ¹²⁵ I Bolton-Hunter reagent. (See, e.g., Bolton etal. (1973) Biochem. J. 133:529.) Candidate molecules previously arrayedin the wells of a multi-well plate are incubated with the labeled NITE,washed, and any wells with labeled NITE complex are assayed. Dataobtained using different concentrations of NITE are used to calculatevalues for the number, affinity, and association of NITE with thecandidate molecules.

XI. Functional Assays

NITE function is assessed by expressing the sequences encoding NITE atphysiologically elevated levels in mammalian cell culture systems. cDNAis subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPORT™ (Life Technologies, Gaithersburg, Md.) and pCR™ 3.1(Invitrogen, Carlsbad, Calif.), both of which contain thecytomegalovirus promoter. 5-10 μg of recombinant vector are transientlytransfected into a human cell line, preferably of endothelial orhematopoietic origin, using either liposome formulations orelectroporation. 1-2 μg of an additional plasmid containing sequencesencoding a marker protein are co-transfected. Expression of a markerprotein provides a means to distinguish transfected cells fromnontransfected cells and is a reliable predictor of cDNA expression fromthe recombinant vector. Marker proteins of choice include, e.g., GreenFluorescent Protein (GFP) (Clontech, Palo Alto, Calif.), CD64, or aCD64-GFP fusion protein. Flow cytometry (FCM), an automated, laseroptics-based technique, is used to identify transfected cells expressingGFP or CD64-GFP, and to evaluate properties, for example, theirapoptotic state. FCM detects and quantifies the uptake of fluorescentmolecules that diagnose events preceding or coincident with cell death.These events include changes in nuclear DNA content as measured bystaining of DNA with propidium iodide; changes in cell size andgranularity as measured by forward light scatter and 90 degree sidelight scatter; down-regulation of DNA synthesis as measured by decreasein bromodeoxyuridine uptake; alterations in expression of cell surfaceand intracellular proteins as measured by reactivity with specificantibodies; and alterations in plasma membrane composition as measuredby the binding of fluorescein-conjugated Annexin V protein to the cellsurface. Methods in flow cytometry are discussed in Ormerod, M. G.(1994) Flow Cytometry, Oxford, New York, N.Y.

The influence of NITE on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding NITEand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding NITE and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

XII. Production of NITE Specific Antibodies

NITE substantially purified using polyacrylamide gel electrophoresis(PAGE) (see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the NITE amino acid sequence is analyzed using LASERGENE™software (DNASTAR Inc.) to determine regions of high immunogenicity, anda corresponding oligopeptide is synthesized and used to raise antibodiesby means known to those of skill in the art. Methods for selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions are well described in the art. (See, e.g., Ausubelsupra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anApplied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistryand coupled to KLH (Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccininide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XIII. Purification of Naturally Occurring NITE Using Specific Antibodies

Naturally occurring or recombinant NITE is substantially purified byimmunoaffinity chromatography using antibodies specific for NITE. Animmunoaffinity column is constructed by covalently coupling anti-NITEantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing NITE are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof NITE (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/NITE binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and NITEis collected.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 3    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 429 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: OVARTUT01              (B) CLONE: 2257452    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    - Met Arg Val Ala Leu Ser Glu Pro Gln Pro Gl - #u Arg Arg Phe Phe Arg    #                15    - Arg Gly Trp Val Thr Phe Asp Arg Ser Val As - #n Ile Lys Glu Ile Cys    #            30    - Trp Asn Leu Gln Asn Ile Arg Leu Arg Glu Cy - #s Glu Leu Ser Pro Gly    #        45    - Val Asn Arg Asp Leu Thr Arg Arg Val Arg As - #n Ile Asn Gly Ile Thr    #    60    - Gln His Lys Gln Ile Val Arg Asn Asp Ile Ly - #s Leu Ala Ala Lys Leu    #80    - Ile His Thr Leu Asp Asp Arg Thr Gln Leu Tr - #p Ala Ser Glu Pro Gly    #                95    - Thr Pro Pro Leu Pro Thr Ser Leu Pro Ser Gl - #n Asn Pro Ile Leu Lys    #           110    - Asn Ile Thr Asp Tyr Leu Ile Glu Glu Val Se - #r Ala Glu Glu Glu Glu    #       125    - Leu Leu Gly Ser Ser Gly Gly Ala Pro Pro Gl - #u Glu Pro Pro Lys Glu    #   140    - Gly Asn Pro Ala Glu Ile Asn Val Glu Arg As - #p Glu Lys Leu Ile Lys    145                 1 - #50                 1 - #55                 1 -    #60    - Val Leu Asp Lys Leu Leu Leu Tyr Leu Arg Il - #e Val His Ser Leu Asp    #               175    - Tyr Tyr Asn Thr Cys Glu Tyr Pro Asn Glu As - #p Glu Met Pro Asn Arg    #           190    - Cys Gly Ile Ile His Val Arg Gly Pro Met Pr - #o Pro Asn Arg Ile Ser    #       205    - His Gly Glu Val Leu Glu Trp Gln Lys Thr Ph - #e Glu Glu Lys Leu Thr    #   220    - Pro Leu Leu Ser Val Arg Glu Ser Leu Ser Gl - #u Glu Glu Ala Gln Lys    225                 2 - #30                 2 - #35                 2 -    #40    - Met Gly Arg Lys Asp Pro Glu Gln Glu Val Gl - #u Lys Phe Val Thr Ser    #               255    - Asn Thr Gln Glu Leu Gly Lys Asp Lys Trp Le - #u Cys Pro Leu Ser Gly    #           270    - Lys Lys Phe Lys Gly Pro Glu Phe Val Arg Ly - #s His Ile Phe Asn Lys    #       285    - His Ala Glu Lys Ile Glu Glu Val Lys Lys Gl - #u Val Ala Phe Phe Asn    #   300    - Asn Phe Leu Thr Asp Ala Lys Arg Pro Ala Le - #u Pro Glu Ile Lys Pro    305                 3 - #10                 3 - #15                 3 -    #20    - Ala Gln Pro Pro Gly Pro Ala Gln Ile Leu Pr - #o Pro Gly Leu Thr Pro    #               335    - Gly Leu Pro Tyr Pro His Gln Thr Pro Gln Gl - #y Leu Met Pro Tyr Gly    #           350    - Gln Pro Arg Pro Pro Ile Leu Gly Tyr Gly Al - #a Gly Ala Val Arg Pro    #       365    - Ala Val Pro Thr Gly Gly Pro Pro Tyr Pro Hi - #s Ala Pro Tyr Gly Ala    #   380    - Gly Arg Gly Asn Tyr Asp Ala Phe Arg Gly Gl - #n Gly Gly Tyr Pro Gly    385                 3 - #90                 3 - #95                 4 -    #00    - Lys Pro Arg Asn Arg Met Val Arg Gly Asp Pr - #o Arg Ala Ile Val Glu    #               415    - Tyr Arg Asp Leu Asp Ala Pro Asp Asp Val As - #p Phe Phe    #           425    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1468 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: OVARTUT01              (B) CLONE: 2257452    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    - CCAACATCTC CCGGGCCGAG ATCATCTCCC TTTGTAAAAG GTACCCAGGC TT - #TATGCGGG      60    - TGGCGCTCTC AGAGCCCCAG CCAGAGAGGA GGTTTTTCCG TCGTGGCTGG GT - #GACCTTCG     120    - ACCGCAGTGT TAACATTAAA GAGATCTGTT GGAACCTGCA GAACATCCGT CT - #CCGGGAGT     180    - GTGAGCTGAG CCCTGGTGTG AACAGGGACC TGACCCGGCG CGTTCGCAAC AT - #CAACGGCA     240    - TCACCCAGCA CAAGCAGATT GTGCGCAACG ACATCAAGCT GGCGGCCAAG CT - #GATCCACA     300    - CGCTGGATGA CAGGACACAG CTTTGGGCCT CAGAACCAGG GACGCCTCCC CT - #GCCCACGA     360    - GCCTGCCCTC GCAAAACCCG ATCTTGAAGA ATATCACCGA CTACCTGATC GA - #GGAAGTAA     420    - GCGCCGAGGA GGAGGAGCTG CTGGGGAGCA GCGGGGGCGC TCCTCCTGAG GA - #GCCTCCTA     480    - AGGAAGGGAA CCCGGCAGAG ATCAACGTGG AGCGGGATGA GAAGTTGATT AA - #GGTCTTGG     540    - ACAAGCTCCT CCTTTACCTG CGCATCGTGC ATTCCTTGGA TTATTACAAC AC - #CTGTGAGT     600    - ACCCCAACGA GGACGAGATG CCCAATCGCT GTGGGATCAT CCACGTTCGG GG - #GCCCATGC     660    - CACCCAACCG CATCAGTCAC GGGGAAGTGC TGGAGTGGCA GAAGACTTTT GA - #GGAGAAGC     720    - TCACGCCGTT GCTGAGTGTG CGGGAGTCAC TCTCAGAGGA AGAGGCCCAG AA - #GATGGGGC     780    - GCAAAGACCC AGAGCAGGAA GTGGAGAAGT TCGTCACCTC CAACACGCAG GA - #ACTGGGCA     840    - AGGATAAGTG GCTGTGTCCT CTCAGTGGCA AGAAATTCAA GGGTCCTGAG TT - #TGTGCGCA     900    - AACATATCTT CAACAAGCAT GCAGAGAAAA TTGAGGAAGT GAAAAAGGAA GT - #CGCGTTTT     960    - TTAACAACTT CCTCACTGAT GCTAAGCGCC CAGCTCTGCC TGAGATCAAG CC - #AGCCCAGC    1020    - CACCTGGCCC CGCCCAGATA CTCCCCCCAG GTTTGACCCC AGGACTCCCC TA - #CCCACACC    1080    - AGACTCCCCA GGGCCTGATG CCCTATGGTC AGCCCCGGCC CCCGATCTTG GG - #CTATGGAG    1140    - CTGGTGCTGT CCGCCCTGCA GTCCCCACAG GAGGCCCTCC ATACCCCCAT GC - #CCCGTATG    1200    - GTGCTGGTCG AGGGAACTAT GATGCCTTCC GAGGCCAGGG AGGTTATCCT GG - #GAAACCTC    1260    - GCAACAGGAT GGTTCGTGGA GACCCAAGGG CCATTGTGGA ATATCGGGAC CT - #GGATGCCC    1320    - CAGACGATGT TGATTTCTTT TGAGCCGTCC CCCGTTCCTC AGTCCTGTAT CA - #TCCATACT    1380    - TGTACTACCT TGTCCTATGA AGCTCTGAGA ATTTTTTGTA CGATCAGCCT TA - #CTGCTAAT    1440    #           1468   GGAA AAAAAAAA    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 225 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -    (vii) IMMEDIATE SOURCE:              (A) LIBRARY: GenBank              (B) CLONE: 1127863    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    - Met Pro Asn Arg Cys Gly Ile Ile His Val Ar - #g Gly Pro Met Pro Pro    #                15    - Asn Arg Ile Ser His Gly Glu Val Leu Glu Tr - #p Gln Lys Thr Phe Glu    #            30    - Glu Lys Leu Thr Pro Leu Leu Ser Val Arg Gl - #u Ser Phe Leu Arg Lys    #        45    - Arg Pro Arg Arg Trp Val Val Lys Thr Glu Gl - #n Glu Val Glu Lys Phe    #    60    - Val Thr Ser Asn Thr Gln Glu Leu Gly Lys As - #p Lys Trp Leu Cys Pro    #80    - Leu Ser Gly Lys Lys Phe Lys Gly Pro Glu Ph - #e Val Arg Lys His Ile    #                95    - Phe Asn Lys His Ala Glu Lys Ile Glu Glu Va - #l Lys Lys Glu Val Ala    #           110    - Phe Phe Asn Asn Phe Leu Thr Asp Ala Lys Ar - #g Pro Ala Leu Pro Glu    #       125    - Met Lys Pro Ala Gln Pro Pro Gly Pro Ala Gl - #n Ile Leu Pro Pro Gly    #   140    - Leu Thr Pro Gly Leu Pro Tyr Pro His Gln Th - #r Pro Gln Gly Leu Met    145                 1 - #50                 1 - #55                 1 -    #60    - Pro Tyr Gly Gln Pro Ala Leu Pro Ile Phe Gl - #y Leu Trp Ser Trp Cys    #               175    - Cys Pro Pro Cys Arg Ser Gln Gln Glu Gly Le - #u His Ile Pro Met Val    #           190    - Arg Met Ala Leu Gly Ala Gly Thr Met Thr Le - #u Phe Glu Ala Lys Glu    #       205    - Asp Ile Leu Gly Asn Leu Gly Thr Gly Trp Ph - #e Glu Glu Thr Arg Gly    #   220    - Pro    225    __________________________________________________________________________

What is claimed is:
 1. An isdolated and purified polynucleotide encodinga polypeptide sequence comprising of amino acid sequence of SEQ ID NO:1.2. An isolated and purified polynucleotide which hybridizes underhybridization conditions of 250 mM NaCl. 25 mM trisodium citrate, 1%SDS, 50% formamide, and 200 μg/ml ssDNA at 42° C. and wash conditions of15 mM NaCl, 1.5 mM trisodium citrate and 0.1% SDS at 68° C. to theentire length of the polynucleotide of claim
 1. 3. An isolated andpurified polynucleotide which is completely complementary to thepolynucleotide of claim
 1. 4. An isolated and purified polynucleotideconsisting of the polynucleotide sequence of SEQ ID NO:2.
 5. An isolatedand purified polynucleotide having a sequence completely complementaryto the polynucleotide sequence of claim
 4. 6. An expression vectorcomprising the polynucleotide sequence of claim
 1. 7. A host cellcomprising the expression vector of claim
 6. 8. A method for detecting apolynucleotide encoding a polypeptide comprising the amino acid sequenceof SEQ ID NO:1 in a biological sample containing nucleic acids, themethod comprising the steps of:a) hybridizing the polynucleotide ofclaim 3 of the nucleic acids of the biological sample, thereby forming ahybridization complex; and b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of the polynucleotide encoding the polypeptide in thebiological sample.
 9. The method of claim 8 wherein the nucleic acids ofthe biological sample are amplified by the polymerase chain reactionprior to the hybridizing step.